It has widely known that a drug is enclosed in fine particles to enhance the effect of the drug, and that the fine particles include, for example, liposome, fat emulsion, etc. Their clinical applications are carried out mainly by injections, particularly by intravascular administration. The fine particles administered to blood vessel have been known to interact with blood components and, as a result of the interaction, the fine particles themselves or the drug is destructed (disintegrated), or the fine particles are opsonized whereupon they are removed from the blood (they are removed as an extraneous substance by a reticuloendothelial system). In order to prevent that removal, modification of liposome with polyethylene glycol, for example, has been studied [Stealth Liposomes, ed. by D. D. Lasic and F. Martin, CRC Press Inc., Florida, 93-102 (1995)].
Further, in order to deliver a nucleic acid such as oligonucleotide, DNA and RNA to target cells, a complex of a nucleic acid with liposome comprising lipid containing cationic lipid (hereinafter, referred to as cationic lipid liposome), a basic polymer such as poly-L-lysine and polyamideamine have been frequently used. However, it has been known that, when a complex of cationic lipid liposome with a nucleic acid is intravenously administered, it is quickly distributed from blood to liver, lung, etc. [Biochim. Biophys. Acta, 1281, 139-149(1996); J. Controlled Release, 41, 121-130(1996)]. On the other hand, S. Li, et al. has reported that, when mouse serum is contacted with a cationic lipid liposome/DNA complex, an increase in the size of the complex, aggregation, disintegration of liposome, and release and disintegration of DNA has taken place [Gene Therapy, 5, 930-937 (1998); Gene Therapy, 6, 585-594 (1999)]. In order to solve those problems, modification of cationic lipid liposome with polyethylene glycol was studied, and O. Meyer, et al. prepared a complex of oligodeoxynucleotide (ODN) with cationic lipid liposome containing polyethylene glycol phosphatidylethanolamine (PEG-PE) [J. Biol. Chem., 273, 15621-15627 (1998)]. However, when it was contacted with a 50% aqueous solution of human plasma for 4 hours, 35% of ODN were dissociated. In order to reduce the dissociation, D. Stuart and T. Allen previously dissolved the cationic lipid liposome in chloroform, mixed the resulting solvent with an aqueous ODN solution and methanol, and transferred a cationic lipid liposome/ODN complex to chloroform layer, and subjecting to centrifugal separation. They further took out the chloroform layer, added thereto PEG lipid, neutral lipid and water to form W/O emulsion. They have tried to enclose ODN inside the liposome completely by forming the W/O emulsion in a manner similar to a reverse phase evaporation method of F. Szoka, et al. [Biochim. Biophys. Acta, 1463, 219-229(2000)]. In recent years, however, the use of chloroform is not considered to be desirable in view of safety. Further, D. McPhail, et al. prepared a vesicle-in-vesicle where chitosan vesicle is placed in liposome by adding a vesicle suspension (chitosan vesicle) of palmitoylchitosan and cholesterol to a thin layer of egg yolk phosphatidylcholine and cholesterol [Int. J. Pharmaceutics, 200, 73-86 (2000)]. However, there is no description about the enclosing efficiency and, when guessed from the preparation method, the enclosing efficacy was presumed to be as low as about a few percent, which is presumed to cause a problem in its practical use. From such viewpoints as well, convenient and highly efficient enclosure of fine particles by closed vesicle is very useful when application to medical treatment is aimed.
Further, there are some cases where many peptides and proteins which are useful in medical care are quickly decomposed in living body by enzyme or the like or are removed from living body as a result of generation of an antibody by frequent administrations, whereby their effect is no longer exerted. Therefore, with an object of enhancing the stability of those peptides and proteins in living body, it has been attempted to enclose them into liposome. As the means of enclosing them into liposome, there have been known, for example, a liposome preparation method by Bangham, et al. [J. Mol. Biol., 13, 238 (1965)], an ethanol injection method [J. Cell Biol., 66, 621 (1975)], a French press method [FEBS Lett., 99, 210 (1979)], a freeze-thaw method [Arch. Biochem. Biophys., 212, 186 (1981)], a reverse phase evaporation method [Proc. Natl. Acad. Sci. USA, 75, 186 (1981)], a pH gradient method (Japanese Patent No. 2,572,554; Japanese Patent No. 2,659,136; etc.) and the like. For low-molecular compounds, a pH gradient method is appropriate and improved method thereof has been devised as well. However, with regard to peptides and proteins, invariably efficient enclosing has not been achieved yet and, in the case of fluorescence-labeled insulin, it was enclosed to an extent of about 5 to 40% but no insulin was enclosed at all [Int. J. Pharmaceutics, 179, 85-95 (1999)]. In order to enhance the therapeutic effect by peptides and proteins, it is in demand to develop a method whereby peptides and proteins are efficiently enclosed within closed vesicles.